Sepsis
Sepsis is a systemic inflammatory response to an infection or insult (Aird, 2003; Dremsizov et al., 2004). Severe sepsis is a serious public health concern in the United States. It is a common diagnosis among critically ill patients and carries a high mortality rate. However, the pathophysiology of sepsis is not well understood (Riedemann et al., 2003) and afflicted patients deteriorate rapidly as a result of the progressive failure of multiple organs. The bulk of recent investigations suggest that it results from uncontrolled responses of the immune and inflammatory systems that are clinically manifested by an acute systemic inflammatory reaction syndrome (SIRS) (Tetta et al., 2005). In sepsis, hyperactivation of the immune response leads to the excessive production of various proinflammatory cytokines and cellular injury (Pinsky, 2004).
LPS
Sepsis can be caused by a non-infectious or infectious insult, including Lypolysaccharide (LPS or endotoxin). LPS is a component of the outer cell membrane of Gram-negative bacteria that can initiate a parallel cascade of events that contribute to the clinical manifestations of sepsis (Alexander and Riestschel, 2001; Dauphinee and Karsan, 2006). For example, in vitro and in vivo studies have shown that LPS interact with cells of the immune system through antigen recognition pattern and with inflammatory cells such as leukocytes, platelets and endothelial cells. Interaction between the cells of the immune and inflammatory systems leads to several responses including the activation of downstream signaling pathways that promote posttranscriptional changes in cell function; up-regulation of cell adhesion molecules that promote interaction between endothelial cells leukocytes and platelets; and increased expression of procoagulants. These responses are maintained by a continuous recruitment and activation of leukocytes and platelets to the site of endothelial injury. One of the consequences is the formation of microthombi. The endothelium shifts from an anticoagulant surface to a procoagulant surface. Activated endothelium expresses tissue factor, releases Von Willebrand factor (VWF), decreases the expression of endothelial protein C receptor (EPCR) and natural anticoagulants such as thrombomodulin (TM) (Li et al., 2005; Iwaki et al., 2005). Endothelial cells (EC) are also greatly responsible for an uncontrolled inflammatory response (Aird, 2003; Peters et al., 2003; Chen and Lopez, 2005). They can be activated directly by LPS or by a number of other mechanisms observed in patients with sepsis such as complement, cytokines, chemokines, coagulation factors, fibrin, activated platelets and activated leukocytes. The result is a self feeding enhancement of the inflammatory response that if uncontrolled becomes irreversible. The clinical manifestation is a rapid deterioration of the patient with progressive failure of multiple target organs and death (Guidet et al., 2005; O'Brien et al., 2005). There are several markers of endothelial cell activation (Meisner, 2005), VWF being one of the most used (Schorer et al., 1987).
VWF
Mature VWF consists of a 2,050-residue polypeptide that contains multiple copies of A, B, C and D type domains, arranged in the order D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (cystine knot) (FIG. 1) (Bonthron et al., 1986; Verweij et al., 1986). Pro-VWF subunits dimerize through disulfide bonds near their carboxyl termini. These pro-VWF dimers form disulfide bond between N-terminal D3 domains and C-terminal CK domains, generating VWF multimers that may contain more than 80 subunits and exceed 20 million daltons (Girma et al., 1987). VWF is synthesized and secreted by endothelial cells into plasma and the vascular subendothelium (Sussman and Rand, 1982). VWF is also stored in and secreted from the α-granules of megakaryocytes and platelets (Nachman et al., 1977). VWF has a significant physiological relevance in hemostasis, thrombosis and inflammation. It interacts with coagulation factor VIII, platelet glycoproteins (GP) Ib/IX, GPIIb/IIIa, fibrin, and collagen (Koppelman et al., 1996; Beacham et al., 1992; Keuren et al., 2004).
As shown in FIG. 1, the central portion of the VWF subunit contains a triplicate repeat sequence, or A domain, that has been identified in other proteins (Whittaker and Hynes, 2002). Investigators have established that the VWF-A1 domain contains binding sites for GPIb/IX, heparin, cell surface sulfatides and collagen types I, III and VI (Vasudevan et al., 2000; Cruz et al., 1993; Perrault et al., 1999; Sobel et al., 1992; Christophe et al., 1991; Borthakur et al., 2003; Mazzucato et al., 1999; Hoylaerts et al., 1997; Morales et al., 2006). Furthermore, investigators have established that the highly homologous VWF-A3 domain, which does not interact with platelets, binds to collagen fibrils, types I and III (Cruz et al., 1995; Lankhof et al., 1996).
The A2 Domain of VWF
The VWF-A2 domain (amino acid residues 1480-1673) (Verweij et al., 1986; Bonthron et al., 1986), is functionally important because it contains the cleavage site for the enzyme ADAMTS-13, a metalloproteinase that controls the size of VWF multimers. In an inflammatory environment, endothelial cells secrete ultralarge multimers of VWF (ULVWF) that constitutively bind platelet GPIb (Moake, 2002) and proficiently recruit platelets to the site of endothelial injury. ADAMTS-13 limits the size of the VWF multimers and prevents spontaneous formation of microthrombi (Fujikawa et al., 2001; Zheng et al., 2001). The site of cleavage is located between the residues Y1605 and M1606 of the A2 domain, (Furlan et al., 1996; Tsai, 1996). Very recently, investigators have expressed in bacteria the VWF-A2 domain for use as a substrate in assays to measure the activity of ADAMTS-13 in plasma (Cruz et al., 2003; Kokame et al., 2004; Whitelock et al., 2004).
VWF and Microvascular Thrombosis
Under healthy conditions VWF is constitutively secreted as high molecular weight multimers into plasma or subendothelium. However, in conditions associated with SIRS, such as in endotoxemia, endothelial cells are activated and release ULVWF from intracellular storage granules (i.e. Weibel-Palade bodies)(van Mourik et al., 2002). As mentioned earlier, ULVWF multimers make a strong bond with the platelet receptor GPIbα, facilitating local accumulation of platelets and formation of thrombi (Dong et al., 2002; Arya et al., 2002).
The release of ULVWF can be induced on human umbilical vein endothelial cells (HUVEC) by a variety of factors that are present in sepsis such as inflammatory cytokines, thrombin, histamine, leukocyte elastase, high shear, hypoxia and endotoxin (LPS) (Aird, 2003; Dong et al., 2002; Gimbrone et al., 2000; Zeuke et al., 2002). In fact, a study showed that inflammatory cytokines such as interleukin (IL)-8 and tumor necrosis factor-alpha (TNFα), induced the release of ULVWF (Bernardo et al., 2004). This newly secreted ULVWF binds to platelets, activating more platelets that in turn activate the endothelial cells. This process, together with the activation of the coagulation cascade, leads to the generation of thrombin and the formation of fibrin. Clinically, these interactions are translated into a thrombotic and hypercoagulable tendency.
The present invention provides novel solutions for long-felt needs in the art to treat medical conditions that are treatable by the A2 domain of VWF, such as sepsis, systemic inflammatory reaction syndrome, and/or thrombosis, for example.